The fungus, Aspergillus niger, secretes several glycoprotein enzymes into its growth medium, many of which have been purified and characterized. The major goal of the proposed research is to determine the mechanism of secretion of those enzymes and the possible role that the carbohydrate plays. This will be accomplished by incubating the cells for various lengths of time with radiolabelled protein and carbohydrate precursors and then isolating cellular compartments (e.g. mitochondria, ribosomes) to determine the intracellular flow of incorporated label. When such studies are coupled with autoradiography and immunocytology (using peroxidase covalently coupled to immunoglobulin from specific hyperimmune sera) at the electron microscopic leved, the results should show the intracellular pathway followed by secretory proteins. Initial glycosylation will be examined by isolating polysomes after pulse labelling, and by establishing an in vitro glycosylating system, containing smooth microsomal membrane, polysomes and GDP-mannose, which could be stimulated by addition of dolichol phosphate and UDP-N-acetylglucosamine. Also the effects of inhibitors, such as 2-deoxyglucose and tunicamycin, will be assayed both in vitro and in vivo.